【作者】 汤细彪；

【导师】 陈焕春； 吴斌；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2010， 博士

【摘要】 多杀性巴氏杆菌(Pasteurella multocida,Pm)是一种重要的病原菌,对多种动物和人均有致病性,可引起猪发生肺炎和萎缩性鼻炎(Atrophic rhinitis,AR),也是猪呼吸道疾病综合征(porcine respiratory disease complex,PRDC)的重要致病因子之一。Pm现已流行于世界养猪业发达的各个国家和地区,该病原菌可与多种病原协同感染,从而增加猪群呼吸道疾病的发病率和死亡率,并造成严重的经济损失。本研究对Pm的病原流行病学、分离菌株的生物学特性、分子多样性、耐药现状、毒力和免疫原性基因进行研究,为我国Pm的流行病学研究提供理论依据,为临床上猪巴氏杆菌病的诊断、有效防控和相关的基础研究奠定基础。主要研究内容如下：1.多杀性巴氏杆菌在我国部分地区猪群中的流行现状2003-2007年对我国部分地区规模化猪场健康猪群和有肺炎与萎缩性鼻炎疑似症状猪的临床样品进行检测,结果从湖北等16个省、市、自治区2,912份有发病症兆猪的鼻拭子、肺脏、胸腔积液、心血和颈部水肿组织中分离出233株Pm,未能从725份健康猪棉拭子中分离出Pm。在我国猪群中,2003-2007年间Pm的总分离率为8.0%；不同年份Pm的总分离率介于6.4-10.2%之间。应用PCR方法对分离的Pm进行荚膜分型,结果证实,分离株中有92株(39.5%)属A型Pm,128株(54.9%)属D型Pm,1株(0.4%)属B型Pm,12株(5.2%)为未定型分离株。同时对分离的49株Pm进行致病性试验,结果高致病株、中致病株和低致病株分别占受试菌的36.7%(18/49)、49.0%(24/49)和14.3%(7/49)；对分离Pm的产毒素能力进行鉴定,结果11株产毒素多杀性巴氏杆菌(Toxigenic Pasteurella,multocida,T+Pm)均为高致病菌株,占61.1%(11/18)。2.猪源多杀性巴氏杆菌耐药表型与基因型的研究对分离的233株Pm菌株进行20种临床常用抗生素的最小抑菌浓度(MICs)试验,结果表明,分离菌株对阿莫西林、林可霉素、克林霉素、四环素和磺胺类药物不敏感,对大观霉素、卡那霉素、庆大霉素和阿米卡星等氨基糖苷类药物中度敏感；而头孢菌素类、氟苯尼考和氟喹诺酮类药物对Pm分离株表现出很高的活性,能有效抑制该类菌群。多重耐药性分析表明,有217株(93.1%)Pm具有多重耐药性；5耐以上的菌株有127株,占54.7%；流行最广的Pm耐药表型为：AMX+LIN+CLI+ TET+SDM+STX。18种耐药基因的PCR检测证实：耐阿莫西林Pm携带blaTEM的阳性率为80.7%；耐林可酰胺类药物Pm携带ermA、ermC和lnuA的阳性率分别为36.7%、49.8%和24.0%；耐甲氧苄啶类Pm携带dfrA5、dfrA7和dfrA13的阳性率分别为18.5%、32.9%和29.5%；而四环素抗性主要由tet(H)、tet(B)和tet(G)介导；磺胺类抗性主要由sull和sul2介导。分析耐药表型与基因型关系表明,同一种抗生素可由不同的耐药基因介导,多数菌株携带有不同的耐药基因,并显著的具有多重耐药性(P<0.01)。3.猪源多杀性巴氏杆菌的毒力基因型与分布特征研究对Pm的19种毒力相关基因进行PCR检测,结果证实,粘附因子ptfA(93.6%)、fimA(99.1%)、hsf-2(99.1%),转铁蛋白exbB(99.1%)、exbD(99.1%).tonB(97.9%)、hgbA(96.6%)、fur(92.7%),唾液酸酶nanH97.0%)和外膜蛋白ompA(100%). ompH(93.1%)、oma87(94.4%).plpB(99.1%)的流行与检出率较高,几乎所有的猪源Pm均携带这些毒力因子；而毒力因子pfha,tadD,toxA和pmHAS在猪源Pm菌株中的检出率均低于50%。不同的毒力因子与荚膜血清型具有一定的相关性,如tadD显著的分布于A型Pm(P<0.001)；hsf-1和nanB显著的分布于D型Pm(P<0.001)；hgbA和fur主要分布于A型和D型Pm(P<0.01)；pfha和pmHAS显著的分布于A型和未定型Pm(P<0.001)；且试验中不同地区分离的11株T+Pm的毒素toxA基因也仅分布于D型。4.猪源多杀性巴氏杆菌分离株的分子多样性研究采用RAPD,(GTG)5-PCR和Eric-PCR分析方法,对从我国不同地区分离的233株猪源Pm进行核酸分子分型和多样性分析。DNA指纹谱型表明RAPD方法的分辨率为39.48%,多态性条带的百分率为73.96%,可将受试的Pm分为92个基因型,其中有11个(比例为12.0%)为优势谱型。在相似系数为60%时,Pm分离株可被分为32个簇,其中最大的1簇含有A型Pm 20株,D型Pm 22株,B型和未定型Pm各1株,且遗传背景相关的16株海南省分离菌聚集在该簇;11株T+Pm也聚集在该簇。(GTG)5-PCR的分辨率为49.36%,多态性条带的百分率为88.13%,可将受试的Pm分为115个基因型,其中优势谱型有7个(比例为6.08%)。在相似系数为60%时,Pm分离株可被分为68个簇。Eric-PCR指纹分析方法的分辨率为24.46%,多态性条带的百分率为73.46%,可将受试的Pm分为57个基因型,其中优势谱型有11个(比例为19.03%)。在相似系数为60%时,Pm分离株可被分为36个簇。综上分析表明,我国不同地区分离的猪源Pm的核酸基因组存在显著的多样性,同时证明了(GTG)5-PCR和RAPD扩增的DNA指纹比Eric-PCR图谱具有更强的多样性。5.猪产毒多杀性巴氏杆菌毒素toxA基因的分子特征与致病性研究为分析我国产毒多杀性巴氏杆菌T+Pm地方分离株的分子特征、遗传背景及毒力特性,对分离的11株T+Pm的毒素toxA基因进行了克隆和测序。结果表明,toxA基因编码的毒素PMT是一个高度保守的蛋白,其核苷酸间的同源性达99%以上,其氨基酸的同源性达98.5%以上,且该基因在911-912和2323-2324核苷酸位点发生点突变的频率较高,达到23.8%(5/21)。对toxA基因的分子进化树分析表明,我国地方T+Pm分离株可分为2个亚群,且菌株间的亲缘关系或遗传背景较近,表现出区域相关性,与韩国、台湾、美国等分离株同属一个进化分支。对小鼠的LD5o试验证实,T+Pm对小鼠具有高致病性,且分离株HN-13表现为强毒力,其对小鼠的LDso为4.25×103CFU。将HN-13株人工感染试验猪,建立T+Pm的感染模型,结果HN-13不但可复制出PAR的临床症状,且该菌株可引起猪肺炎。免疫组织化学分析证实,HN-13株感染猪的急性期,机体中大量的嗜中性白细胞发生局部聚集,以抵抗病原菌的侵入,在肺泡内同时也出现了巨噬细胞吞噬感染的T+Pm,并造成巨噬细胞的坏死和髓样病变。6.毒素PMT的N-端和C-端蛋白的生物学活性及免疫原性研究将5个toxA基因片段N1518、C2345、N3172、N3388和C2115分别克隆到合适的原核表达载体pET-28a(b,c)系统,其中pET28a-N1518和pET28b-C2115在大肠杆菌成功表达,获得大小分别为57kDa和78kDa的融合蛋白rPMT-N和rPMT-C,Western blot检测证实这两种表达产物均具有反应原性。分别以200μg rPMT-N和rPMT-C对小白鼠进行体内生物学活性试验,结果两种表达蛋白均不能致死小白鼠；体外细胞毒性试验证实896ng/ml的rPMT-N能使Vero细胞发生病变,而rPMT-C对Vero细胞无明显毒性作用。将rPMT-N和rPMT-C制成亚单位疫苗,同时设天然PMT及无菌PBS对照组,间隔2周分2次免疫小白鼠和仔猪。结果表明rPMT-N、rPMT和天然PMT试验组能诱导小鼠产生较高滴度的毒素抗体,而rPMT-C诱导的毒素抗体滴度较低；对仔猪免疫效力试验证实,rPMT-N、rPMT、rPMT-C和天然PMT均能诱导机体产生针对PMT的毒素抗体,但用1×1010CFU的HN-13株对各组试验仔猪进行鼻腔感染试验后,rPMT-C提供的保护力较rPMT-N、rPMT和天然PMT组低。综上试验表明,rPMT-N具有良好的生物学活性和免疫原性,可作为PAR疫苗添加成分,显示了良好的应用前景。更多还原

【Abstract】 Pasteurella multocida is an important pathogenic bacterium, which is responsible for various infections in humans and animals. The main diseases associated with this organism in pigs are pneumonic pasteurellosis and atrophic rhinitis. P. multocida is also a contributory agent in porcine respiratory disease complex, a multifactorial disease state increasingly problematic for swine producers. Although the primary disease is important, what’s more significant is the fact that this bacteria predisposes to colonization and disease with other viral and bacterial pathogens, resulting in higger morbidity and mortality. This study focused on the pathogen epidemiology, bio-characteristics, antibiograms, virulence genetypes, the pathogenicity of P. multocida, immunogenicity genes, diagnostic method, and two fragments of recombinant subunit P. multocida toxin (rsPMT) were constructed for evaluation as candidate vaccines against progressive atrophic rhinitis (PAR) of swine. The results are as follows.1. Isolation and identification of P. multocida from pigs and pathogen epidemiology of pasteurellosis in ChinaThe presence of P. multocida in clinical specimens from diseased pigs with pneumonia or atrophic rhinitis from 16 provinces in China between 2003 to 2007 were analyzed, and a total of 233 isolates of Pasteurella multocida were obtained from 2,912 cases of clinical respiratory disease in pigs, giving an isolation rate of 8.0%. The isolation rate at different time points (years) were ranged from 6.4 to 10.2%. Serogroup A P. multocida was isolated from 92 cases (39.5%) and serogroup D isolates from 128 cases (54.9%); twelve isolates (5.2%) were untypable. P. multocida was the fourth most frequent pathogenic bacterium recovered from the respiratory tract, after Streptococcus suis, Haemophilus parasuis and Escherichia coli. The pathogenicities of 49 porcine P. multocida strains isolated from different regions of China were evaluated in mice. Based on mortalities occurring during a 7-day postinoculation period,36.7%,49.0% and 14.3% of 49 isolates were characterized as high, intermediate and low in pathogenicity, respectively. Among them, eleven toxigenic strains of P. multocida of serogroup D isolated from 37 samples of nasal swabs and lesioned lungs from pigs with typical clinical signs of atrophic rhinitis were characterized as high in pathogenicity. These results reveal that P. multocida are widely prevalent in China, and suggest such pathogenic bacteria may play an important role in causing respiratory disease of swine. 2. Phenotypic and genotypic characterization of antimicrobial resistance in P. multocida isolatesAntibiograms and relevant genotypes of porcine P. multocida isolates (n=233) recovered between 2003 and 2007 in China were assessed via broth microdilution test, PCR and sequencing. Among the 20 antimicrobials tested, the most prevalent resistance was to amoxicillin, lincomycin, clindamycin, tetracycline and sulfonamides (ranging from 58.0-96.6%), followed by tilmicosin (28.3%) and aminoglycosides (ranging from 12.0-14.2%). The frequency of antimicrobial resistance among P. multocida isolates from swine in China was higher than that reported from other countries, and 93.1% of the isolates showed multiple resistances,54.7% isolates were resistant to more than five. The most common resistance pattern observed among the isolates was multi-resistance to AMX+LIN+CLI+TET+SDM+STX. There was a progressive increase in multiresistance to more than seven antibiotics, from 16.2% in 2003 to 62.8% in 2007. Resistance profiles suggested that cephalosporins, florfenicol and fluoroquinolone were the drugs most likely to be active against P. multocida. For each antimicrobial agent, amoxicillin resistance was primarily mediated by blaTEM (80.7%), lincosamides resistance by ermA (36.7%), ermC (49.8%) and lnuA (24.0%), trimethoprims resistance by dfrA5 (18.5%), dfrA7 (32.9%) and dfrA13 (29.5%), tetracycline resistance by tet(H), tet(B) and tet(G), and sulfadimidine resistance by sull and sul2. Strains that harbored several genes that conferred resistance to the same antimicrobial agent were often significantly (P< 0.01) more multiresistant than others, and there was positive relation between drug resistance and genetypes. These results provide novel insights into the epidemiological characteristics of porcine P. multocida strains in China, and suggest the need for the prudent use of antimicrobial agents in food animals.3. The presence and distribution of virulence genes of P.multocida strains from swineAmong the 233 porcine P. multocida isolates, the 19 virulence gene regions ranged in prevalence from 4.7%(toxA) to 100%(ompA). Multiple adhesions (including ptfA, fimA and hsf-2),all iron acquisition factors (exbB, exbD, tonB, hgbA and fur), nanH, and various outer membrane proteins (ompA, ompH, oma87 and plpB) were found each to occur in over 90% of porcine strains. The VFs pfhA, tadD, toxA and pmHAS were present in<50% of strains each. The various VFs exhibited distinctive associations with serogroups, tadD was significantly associated with serogroup A, hsf-1 and nanB were significantly associated with serogroup D, and hgbA and fur were associated with serogroups A and D. The genes pfhA and pmHAS were less common in serogroup D than in other serogroups. It was noted that the toxA gene, which is involved in the pathogenesis of progressive atrophic rhinitis in pigs, was found in only eleven strains, and it was strictly restricted to strains belonging to capsular serogroup D.4. Molecular diversity of P.multocida isolates from diseased swine in ChinaA total of 233 porcine P. multocida strains isolated from cases of pneumonia and progressive atrophic rhinitis (PAR) were evaluated for the genotypes and genetic diversity using several DNA fingerprinting methods:RAPD, (GTG) 5-PCR and Eric-PCR. The discriminatory index (CI) for RAPD was 39.48%, and 73.96% distinct bands were identified as polymorphic bands. Analysis of DNA banding patterns generated by RAPD revealed the presence of 92 different genotypes, among which 11 genotypes (12.0%) were preponderant profiling. All isolates could be grouped into 32 clusters at a similarity of 60% after dendrogram analysis. The largest lineage contained 44 strains belonging to serogroup A (20 strains), serogroup D (22 strains), serogroup B (1 strain) and untypable (1 strain), respectively. The discriminatory index for (GTG) 5-PCR was 49.36%, and 88.13% distinct bands were identified as polymorphic bands. These isolates could be divided into 115 different genotypes by their (GTG) 5-PCR fingerprints, among which 7 genotypes (6.08%) were preponderant fingerprints. All isolates could be grouped into 68 clusters at a similarity of 60%. The discriminatory index for Eric-PCR profiling was a relatively low value (CI=24.46%), and 73.46% distinct bands were identified as polymorphic bands. These isolates could be divided into 57 different genotypes by their Eric-PCR fingerprints, among which 11 genotypes (19.03%) were preponderant fingerprints. All isolates could be grouped into 36 clusters at a similarity of 60%. In summary, the results revealed there was significant diversity among analysed strains of porcine P. multocida strains, and suggested that (GTG) 5-PCR and RAPD are highly discriminatory techniques for detecting genetic variation and in epidemiological studies of P. multocida.5. Molecular characteristics and the pathogenicity of porcine toxigenic P. multocidaIn order to analysis the molecular characteristics, genetic background and the pathogenicity of toxigenic P. multocida isolates from swine in China, toxA gene from eleven strains were investigated. Their sequences analyzed here indicated that the P.multocida toxin (PMT) coded by toxA gene is a high conservatism protein. The homology of toxA gene nucleotide sequences of all strains hit above 99%, and the homology of amino acid sequences reached to 98.5% above. However, the nucleotides of toxA gene of five strains,have genesised mutation,in the site 911-912 and 2323-2324. The prevalence and genetic evolution were analyzed based on the toxA gene. We found that there are two highly diverse subgroups among all T+Pm isolates in China. By virulence tests, we found that the HN-13 strain of T+Pm showed high virulence, and the 50% lethal dose (LD50) of HN-13 in mice have been tested to be 4.25×103 CFU intra-abdominal infection. Further, the HN-13 intranasal infection model was also established using piglets. The results showed that there testing piglets not only have the clinical symptom of PAR, but also could cause pneumonia. Immunohistochemistry confirmed that neutrophil could be mobilized to counteracting incursive pathogenic bacteria during acute stage, and macrophagocyte could also participate in the mechanism, inducing cellular necrosis and pulpy pathological changes.6. Characteristics and immunogenicity of the N-terminal and C-terminal re-combinants of P.multocida toxinIn this study, five fragments of recombinant subunit P.multocida toxin (PMT) were constructed. Only pET28a-N1518 and pET28b-C2115 could be expressed efficiently in Escherichia coli. The molecular weight of the fusion proteins was 57 kDa and 78 kDa. Western blot confirmed that the two proteins could specifically react with an-tiserum against P.multocida toxin. No mice died after the intraperitoneal administration of these two proteins with the dose of 200μg, but Vero cell was pathologically changed after administration of 896ng/mL rPMT-N. The fusion protein of rPMT-N and rPMT-C was purified, and emulsified with Freund’s adjuvant in equal volumes to get subunit vaccine. Mice immunized with rPMT-N, rPMT containing rPMT-N and rPMT-C, crude PMT only developed high titers of neutralizing antibodies. However, all piglets were challenged intranasally with 1×1010CFU CFU HN-13 strain of T+Pm. The protection efficiency of rPMT-N, rPMT and crude PMT against HN-13 strain were higher than the group immunized with rPMT-C. The data revealed that the fusion protein of rPMT-N had immunogenicity and potential for developing a subunit vaccine against PAR in pigs.更多还原